Long-term storage of animal cells and tissues is of widespread critical importance to the research and biomedical fields. Cryopreservation of cells and tissues is useful, for the long-term storage of cell lines to provide an unchanging population of cells; and the storage of populations of cells for research or medical purposes.
It is widely held that animal cells can be stored indefinitely once they reach liquid nitrogen temperature (−196° C.). It has been well-established, however, that the freezing process itself results in immediate and long-term damage to cells with the greatest damage occurring to cells as they traverse the intermediate zone of temperature (−15° C. to −60° C.) during cooling and thawing (Mazur, Am. J. Physiol., 247:C125-142, 1984). The primary damaging physical events that can occur to cells during the process of freezing include dehydration and intracellular ice crystal formation. During freezing, solute is rejected from the solid phase producing an abrupt change in concentration in the unfrozen portion of solution. A biological cell responds to this perturbation by dehydrating to reach a new equilibrium state between intracellular and extracellular solutions. At high cooling rates, equilibrium cannot be maintained because the rate at which the chemical potential in the extracellular solution is being lowered is much greater than the rate at which water can diffuse out of the cell. The end result of this imbalance is that intracellular ice formation is observed which is lethal to the cell (Toner, J. of Applied Phys., 67:1582-1593, 1990). At low cooling rates, cells are exposed for long periods of time at high subzero temperatures to high extracellular concentrations resulting in potentially damaging high intracellular concentrations (Lovelock, Biochem. Biophys. Acta, 10:414-446, 1953).
There have been attempts in the art to incorporate the process of vitrification into methods of cryopreserving cells. The aim of vitrification is to lower the temperature of a cell suspension while avoiding the formation of ice crystals by the use of viscous or concentrated liquid solutions. This approach is fundamentally different to standard methods of freezing that concentrate more so on carefully controlling the formation of ice crystals Methods incorporating vitrification have shown some promise however recoveries can be poor. Furthermore, the methods are not amenable to automation, and therefore quality control can be difficult. Another problem is that compounds such as polyethylene glycol are required in the vitrification solution. A further problem with vitrification is that the vessels used severely limit the amount of material that can be frozen. Additionally, the commonly used “open straws” do little to avoid the possibility of microbial cross-contamination of the material to be frozen.
The clinical and commercial application of cryopreservation for certain cell types is limited by the ability to recover a significant number of total viable cells that function normally. Significant losses in cell viability are observed in certain primary cell types. Examples of freeze-thaw cellular trauma have been encountered with cryopreservation of hepatocytes (Borel-Rinkes et al., Cell Transplantation, 1:281-292, 1992) porcine corneas (Hagenah and Bohnke, 30:396-406, 1993), bone marrow (Charak et al., Bone Marrow Transplantation, 11:147-154, 1993), porcine aortic valves (Feng et al., Eur. J. Cardiothorac. Surg., 6:251-255, 1992) and human embryonic stem cells (hESCs; http://www.wicell.org/forresearchers, FAQs—Culturing Human ES Cells: FAQs 4 & 8; Reubinoff et al., Human Reprod, 16(10):2187-2194, 2001).
The regulatory requirements for producing clinically acceptable hESCs present unique characteristics and accompanying challenges. For example, for a hESC to be useful in routine therapeutic applications it will be necessary to generate and store cells in a Master Cell Bank from a single hESC source. Compliance will ensure quality assurance and safety towards maximizing clinical efficacy, the primary mandate for the Food and Drug Administration (FDA). A suitable cryopreservation method that satisfies existing and future regulations under Good Tissue Practice (GTP) and Good Manufacturing Practice (GMP) will be essential to the manufacture and use of viable material for cell based therapy. Thus, a standardized procedure with validated components, free from sensitising reagents such as certain animal sera and selected proteins, performed under conditions designed to minimize contamination with adventitious agents, and amenable to high throughput processing for production of large cell banks is a necessary prerequisite.
Cryopreservation protocols typically require the use of cryoprotective agents (“CPAs”) to achieve improved survival rates for animal cells. A variety of substances have been used or investigated as potential additives to enhance survival of cells in the freezing process. Other substances used include sugars, polymers, alcohols and proteins. CPAs can be divided roughly into two different categories; substances that permeate the cell membrane and impermeable substances. One mechanism of protection results from reduction in the net concentration of ionic solutes for a subzero temperature when a CPA is present. This colligative effect is true for all substances that act as a CPA (Fahy, Biophys. J. 32:837-850, 1980). The addition of a CPA however, changes the ionicity of the solution. Both tissues and intact organs can exhibit reduced cellular viability when exposed to sufficiently large step changes in external osmolarity produced by introduction of a freezing solution (Pegg, Cryobiology, 9:411-419, 1972). In addition, long term exposure to even low concentrations of certain CPAs at room temperature is potentially damaging (Fahy, Cryobiology, 27: 247-268, 1990).
Another media component routinely added to freezing media to reduce cell damage and death during freezing and thawing is serum. This additive, however, is highly complex and may add a number of factors (known and unknown), which may interfere with or alter cell function. Other non-permeating protective agents such as ethylene glycol, polyvinyl pyrrolidone (Klebe and Mancuso, In Vitro, 19:167-170, 1983) sucrose, and culture medium (Shier and Olsen, In Vitro Cell Dev. Biol., 31:336-337, 1995), have been studied for their effectiveness as cryoprotective agents for cells with variable results. U.S. Pat. No. 4,004,975 to Lionetti et al. discloses the cryopreservation of leukocytes from centrifuged blood in a solution of hydroxyethyl starch and dimethylsulfoxide. U.S. Pat. No. 5,071,741 to Brockbank and PCT WO 92/08347 to Cryolife, published May 29, 1992, disclose the use of algae-derived polysaccharides such as agarose and alginate in a cryoprotective cell medium. U.S. Pat. No. 5,405,742 to Taylor discloses a solution for use as a blood substitute and for preserving tissue that includes dextran.
PCT WO 95/06068 discloses the use of polysaccharides to improve hematopoietic functions and serve as a radioprotective agent. The use of gum arabic, cherry resin and apricot resin in ewe semen freezing medium is disclosed in Platov et al. (Ovtsevodstvo, 10:38-39, 1980, abstract). Holtz et al. (Proc. Fourth Intern. Symp. Repr. Phys. Fish, 1991) discloses the use of saccharides such as glucose and sucrose in the cryopreservation of trout semen. Hill et al. (J. Lab. Clin. Med., 111:73-83, 1988) discloses the use of arabinogalactan to obtain washed murine platelets by centrifugation. Maisse (Aquat. Living Resour., 7:217-219, 1994) discloses a study of the effect of carbohydrates such as glucose and maltose on the cryopreservation of trout sperm. Isotonic sucrose in combination with calf serum has been used in a medium for the cryopreservation of animal cells (Shier and Olsen, In Vitro Cell. Dev. Biol., 31:336-337, 1995).
Even in consideration of the many years of research in the field of cryopreservation, there is still a need in the art for alternative and improved methods and compositions for freezing cells. There is a special need for methods suitable for hESCs. hESCs have the potential to develop into all or nearly all of the more than 200 cell types in the human body. They have much therapeutic value for treating disease and regenerating damaged tissues and organs. Their clinical potential, however, hinges on their ability to be easily and reliably passaged, frozen, transported, stored and used.
Like many cells used in biomedical research, embryonic stem cells are currently stored and transported in a cryopreserved state in a liquid nitrogen bath. When researchers thaw the cells for use in the lab, however, less than 1% remain viable. The few surviving cells must be placed in culture and painstakingly tended to for weeks before new colonies are abundant enough to be useful for experiments or therapy. The low survival rate makes working with the stem cells time and labour intensive. Furthermore because so few cells survive freezing, natural selection may be altering cell lines in unknown and undesired ways.
In addition, to satisfy existing and future requirements for GTP and GMP and provide quality assurance for the therapeutic utility of stem cells, there is a need for a cryopreservation method with validated components, free from sensitising reagents such as certain animal sera and selected proteins, performed under conditions designed to minimize contamination with adventitious agents, and amenable to high throughput processing for production of cell banks.
It is therefore an aspect of the invention to overcome a problem of the prior art to provide improved methods for the cryopreservation of stem cells, ensuring suitable post-freeze/thaw cell viability and cell quality for the therapeutic utility of stem cells.
The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of this application.